1. Field of the Invention
The present invention relates to microbiological industry, specifically to a method for producing compounds derived from carbamoylphosphate. More specifically, the present invention concerns the using of new feedback-resistant enzymes involved in arginine and pyrimidine biosynthesis pathways of E. coli strains producing compounds derived from carbamoylphosphate, such as arginine, citrulline and pyrimidine derivatives including orotic acid, uridine, uridine 5′-monophosphate (UMP), cytidine and cytidine 5′-monophosphate (CMP).
2. Description of the Related Art
The carbamoylphosphate synthetase (CPSase) of E. coli catalyzes the complex synthesis of carbamoylphosphate (CP) from bicarbonate, glutamine and two molecules of Mg-ATP, with the release of glutamate, phosphate, and two Mg-ADP [Meister A., Advan. Enzymol. Mol. Biol., vol. 62, p. 315–374, 1989]. The synthesis of CP is intermediate for two biosynthetic pathways, namely those of pyrimidine nucleotides and arginine. In the first pathway, CP is coupled to aspartate carbamoyltransferase (ATCase), resulting in formation of orotate in two steps. Orotate is an important metabolic intermediate for the biosynthesis of pyrimidine derivatives, including pyrimidines, such as uracil; pyrimidine nucleosides, such as orotidine, uridine, and cytidine; and pyrimidine nucleotides, such as orotidine 5′-monophosphate (OMP), UMP, and CMP. It was shown that the presence of orotate in a culturing medium during fermentation of the wide scope of bacteria assists measurably in the production and accumulation of pyrimidine derivative, namely, uracil (U.S. Pat. No. 3,214,344). In the second pathway, CP is coupled to ornitine via ornitine carbamoyltransferase (OTCase), constituting the sixth step (starting from glutamate) in the arginine biosynthetic pathway. CPSase is activated by ornitine and IMP (a precursor of purine nucleotides) and inhibited by UMP. Carbamoylphosphate synthetase consists of two subunits. It has been known for coryneform bacteria (EP1026247A1) and for bacteria belonging to the genera Escherichia and Bacillus that those subunits are encoded by carA and carB genes. Transcription of the carAB operon is cumulatively repressed by the end-products of both pathways [Charlier D., et al., J. Mol. Biol., vol. 226, p. 367–386, 1992; Wang H., et al., J. Mol. Biol., vol. 277, p. 805–824, 1998; Glansdorff N., et al., Paths to Pyrimidines, vol. 6, p. 53–62, 1998]. The native E. coli CPSase is a heterodimer composed of a small subunit of 41,270 Da and a large subunit of 117,710 Da, encoded by carA and carB genes respectively. The small subunit catalyzes the hydrolysis of glutamine and is responsible for the transfer of NH3 to the large subunit, where the CP synthesis actually takes place. The large subunit contains the binding sites for the substrates bicarbonate, ammonia, two separate sites for Mg-ATP and a 18 kDa carboxyterminal region which constitutes the regulatory domain [Rubio V., et al., Biochemistry, vol. 30, p. 1068–1075, 1991; Cervera J., et al., Biochemistry, vol. 35, p. 7247–7255, 1996]. Further, it is suggested that the large subunit has an activity to catalyze solely a synthetic reaction of carbamoylphosphate (Stephen D. Rubino et al., J. Biol. Chem., 206, 4382–4386, 1987).
The crystal structure of an allosterically activated form of CPSase has recently been described [Thoden J., et al., Biochemistry, vol. 36, p. 6305–6316, 1997; Thoden J., et al., Acta Crystallogr. Sec. D., vol. 55, p. 8–24, 1999]. The first three distinct domains in the large subunit labeled as A, B, C are very similar in terms of structure, but the fourth one is entirely different. The D domain (residues 937–1073) is responsible for the binding and allosteric regulation by effectors: IMP, UMP and ornitine. Also it was shown, that two residues, serine 948 and threonine 1042, appear to be crucial for allosteric regulation of CPSase [Delannay S., et al., J. Mol. Biol., vol. 286, p. 1217–1228, 1999]. When serine 948 is replaced with phenylalanine, the enzyme becomes insensitive to UMP and IMP, but still activated by ornitine, although to a reduced extent. The enzyme with T1042I mutation displays a greatly reduced activation by ornitine.
As a rule, the feed back resistance (fbr) phenotype of enzyme arises as a result of the replacing the amino acid residue with another in a single or in a few sites of amino acid sequence and these replacements lead to reducing the activity of enzyme. For example, the replacing of natural Met-256 with each of 19 other amino acid residues in E. coli serine acetyltransferase (SAT) (cyse gene) leads in most cases to fbr phenotype but the mutant SAT proteins do not restore the level of activity of natural SAT [Nakamori S. et al. AEM, vol. 64, p. 1607–1611, 1998]. So, the disadvantage of the mutant enzymes obtained by these methods is the reduced activity of mutant enzymes in comparison with the wild type enzymes.